Automated Assignment in Selectively Methyl-Labeled Proteins
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Xu, Yingqi, Minhao Liu, Peter J. Simpson, et al. “Automated Assignment in Selectively Methyl-Labeled Proteins.” Journal of the American Chemical Society 131, no. 27 (2009): 9480–81. https://doi.org/10.1021/ja9020233.
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This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/ja9020233.
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Abstract
Specific methyl labeling schemes and transverse relaxation optimized spectroscopy (TROSY) has extended the molecular size range for the application of NMR spectroscopy to very large proteins (up to ∼1 MDa). Existing strategies for resonance assignment of methyl groups in large systems are based on NMR spectra recorded on smaller fragments and mutants. This is very time-consuming, and chemical shift changes due to mutation or truncation can often complicate interpretation. We have developed a new automated procedure able to rapidly assign the majority of methyl groups in very large proteins, without recourse to mutagenesis or truncated fragments (http://nmr.bc.ic.ac.uk/map-xs/). We demonstrate the effectiveness of this approach on the 300 kDa, ILV-labeled proteasome (α₇α₇) for which excellent spectra have been previously recorded. Of the observed methyl groups, 99% can be correctly assigned in a matter of minutes without manual intervention.